6alpha, 16alpha-dimethyl-pregnenes



United States Patent 3,004,994 GaJGc-DIMETHYL-PREGNENES Glen E. Arth,Cranford, Roger E. Beyler, Westfield, and Lewis H. Sarett, Princeton,N.J., assignors to Merck & Co., Inc., Rahway, NJ., a corporation of NewJersey No Drawing. Filed Sept. 16, 1957, Ser. No. 683,923 26 Claims.(Cl. 260-39145) This invention is concerned generally with novel steroidcompounds and with processes of preparing the same. More particularly,it relates to novel 6,16-dimethyl-l1-oxygenated-1,4-pregnadiene-l7a,2l-di0l-3,20-dione compounds, and to theprocess of preparing these compounds starting with16-methyl-4-pregnene-17a,21-diol-3,11,20- trione. These novel6,16-dimethyl-1l-oxygenated-1,4- pregnadiene-17a,2l-diol-3,20-dionecompounds possess extremely high anti-inflammatory activity, and areespecially efiective for the treatment of arthritis and related diseasessince they can be administered for their cortisonelike action inextremely low dosage thereby minimizing undesired side effects.

These novel6,16-dimethy1-l1-oxygenated-1,4-pregnadiene-l7a,2l-diol-3,20-dionecompounds, subject of the present invention, may be chemicallyrepresented as follows:

CH: wherein X is hydrogen or halogen, R stands for hydrogen or acyl, andZ is a keto or hydroxy substituent.

These 6,16 dimethyl 11 oxygenated 1,4 pregnadiene-17a,2l-diol-3,20-dionecompounds are prepared by reacting16-methyl-4-pregnene-17a,2l-diol-3,11,20-trione with formaldehyde underacidic conditions to form 170:, 20,20,21 bismethylenedioxy 16a methyl 4pregnene- 3,l1-dione which is reacted with ethylene glycol in thepresence of an acidic catalyst to produce 3-ethylenedioxy- 17 a,20,20,21bismethylenedioxy 16a methyl 5 pregnene-ll-one. The latter compound isreacted with perbenzoic acid, perphthalic acid and the like, therebyforming 3 ethylenedioxy 17a,20,20,21 bismethylenedioxy 5,6 epoxy 16methyl pregnane 11 one which is then reacted with formic acid to producea mixture of 17a,20,20,2l-bismethylenedioxy-16a-methyl-6-tormyloxy-pregnane-S-ol-3,ll-dione and 17a,20,20,21- bismethylenedioxy16cc methyl 5 formyloxy pregnane-6-ol-3,11-dione, which mixture, uponreaction with an aqueous alkali hydroxide solution, is converted to170:,20,20,21 bismethylenedioxy 16c: methyl allopregnane-3,6,ll-trione.The last-named compound is reacted with butanone dioxolane to produce3-ethylenedioxy 170:,20,20,21 bismethylenedioxy 16amethylallopregnane-6,ll-dione which is reacted with a methyl Grignardreagent to form 3-ethylenedioxy-17a,20,20,21- bismethylenedioxy 611,161dimethyl allopregnane- 65 o1 11 one. This 3 ethylenedioxy 17a,20,20,21-bismethyleuedioxy 6cz,16ct dimethyl allopregnane- 6,8-01-11-one isreacted with a dehydrating agent such as thionyl chloride in pyridine toform the corresponding 3 ethylenedioxy 17a,20,20,21 bismethylenedioxy 6,16a-dimethyl-5-pregnene-1l-one, which, upon reaction with p-toluenesulfonic acid monohydrate in acetone is converted to17a,20,20,21-bismethy1enedioxy-6a,16a-di- 3,004,994 Patented Oct. 17,1961 2 methyl-4-pregnene-3,ll-dione; the latter compound is reacted withan aqueous organic acid hydrolyzing agent to form6a,l6a-dimethyl-4-pregnene-l7a,21-di0l-3,11,20- trione which can bereacted with an acylating agent to form the corresponding 21-acylate.Alternatively, the 3 ethylenedioxy 17a,20,20,2l bismethylenedioxy 6,l6a-dimethyl-5-pregnene-1l-one, above-mentioned, can be reacted withlithium aluminum hydride thereby reducing the ll-keto substituent to an11,8-hydroxy radical to produce3-ethylenedioxy-17u,20,20,2l-bismethylenedioxy-6,1Ga-dimethyLS-pregnene-Sllfl-ol which is converted, by reaction withp-toluenesultonic acid monohydrate in acetone, to17a,20,20,2l-bismethylenedioxy-6a,Mix-dimethyl- 4-pregnene-11fl-ol-3cne;the latter compound is reacted with an aqueous organic acid hydrolyzingagent thereby forming 6a,l6a-dimethyl 4 pregnene-l15,17a-2l-trio1-3,20-dione which can be reacted with an acylating agent thereby formingthe corresponding 21-acylate derivative.

This 6m,16ot dimethyl 4 pregnene 1lfi,17a,21- triol-3,20-dione21-acylate is reacted with a dehydrating agent such as methane sulfonylchloride in pyridine, or phosphorus oxychloride dissolved in pyridine,to produce the corresponding 60:, l6a-dimethyl-4,9 1 l )-pregnadiene-17a,21-diol-3,20-dione 21-acylate; the latter compound is reacted withhypobromous acid (e.g. N-bromosuccinimide and perchloric acid) toproduce 9cz-b1'Om0-6cz,16ozdimethyl 4 pregnene 11p,17a,21-trio1 3,20dione 21-acylate which is reacted with anhydrous potassium acetate inethanol to produce 6a,16a-dimethyl-9,1l-epoxy-4-pregnene-17a,21-diol-3,20-dione 21-acylate. This 9,11- epoxide is thenreacted with hydrogen fluoride in tetrahydrofuran to produce 60:,160tdimethyl 9a fluoro- 4-pregnene-1l5,l7a,21-triol-3,20-dione 2l-acylate;this compound is reacted with a hydrolyzing agent to form 6a,16adimethyl 9oz fluoro 4 pregnene 115,170, .2l-triol-3,20-dione, which canbe reacted with an acylating agent to produce6a,16a-dimethyl-9a-fluoro-4-pregnene-l 1B,l7a,21-triol-3,20-dione 2l-acylate.

This 6a,l6a-dimethyl-9a-fiuoro-4-pregnene-11fi,17a,21- triol-3,20-dione21-acylate is reacted with chromium trioxide in pyridine to form6a,l6m-dimethyl-9a-fluoro-4- pregnene 17a,2l diol 3,11,20 trione 21acylate which, upon reaction with a hydrolyzing agent, forms 6:1,16czdimethyl c fluoro 4 pregnene :,21- triol-3,ll-20-trione.

The 60:,16a-di11'16ihY1-1 l-oxygenatedi-pregnene-17a,21- diol-3,20-dionecompounds prepared hereinabove e.g. the 6a,l6a dimethyl 4 pregnene170:,21 diol 3,11,20- trione compounds and the6a,l6a-dimethyl-4-pregnene- 11B,17cc,21-i1'i0l-3,20-di011 compounds, arecontacted with the dehydrogenating activity of Schizomycetesmicroorganisms, or with selenium dioxide, thereby forming thecorresponding 6a,l6u-dimethyl-l1-oxygenated-l,4- pregnadiene-17a,21-dio1-3,20di0116 compound.

The lo rnethyl 4 pregnene-17a;l-diol-3,1'1,20-trione compound used asstarting material in the above-mentioned procedure is convenientlyprepared starting with the known 16-pregnene-3a-ol-11,20-dione 3-acetatein accordance with the following procedure: l6-pregnene-3a-ol-ll,20-dione 3-acetate is reacted with methyl mag nesium iodide inthe presence of cuprous chloride thereby forming16m-methyl-pregnane-3a-ol-l1,20-dione 3-acetate, which is reacted withaqueous methanolic hydrochloric acid to form16a-mcthyl-pregnane-3a-ol-11,20-dione. The latter compound, which is apotent anaesthetic, is reacted with acetic anhydride in the presence ofp-toluene sultonic acid catalyst to form a mixture of enol acetatescontaining l6oa-methyl-17,20-pregnene-3a, 20-diol-ll-one 3,20-diacetate;this mixture, after chromatographic purification to remove any unchangedstarting material, is reacted with perbenzoic acid and the resulting16a-methyl-17a-20-epoxy-pregnane-3a,20-diolll-one 3,20 diacetate ishydrolyzed with methanolic" po- 17a,21-diol-3,11,20-trione 21-acetate isreacted with bro mine in glacial acetic acid-chloroform to produce 4-bromo 16oz methyl pregnane 1741,21 diol 3,11,20- trione, which is thenreacted with semicarbazide to form 160: methyl 4 pregnenc 170:,21 diol 73,11,20 trione 3,20-bis-semicarbazone 21-acetate. The lGa-methyl 4pregnene 1705,21 diol 3,11,20 trione 3,20- bis-semicarbazoneZI-acetateis reacted with potassium bicarbonate or potassium hydroxidein aqueous methanol to form16a-methyl-4-pregnene-170:,21-di0l-3,11,20-trione 3,20-bis-semicarbazonewhich is then hydrolyzed under acid conditions to produce16a-methyl-4-pregnene- 17a,21 diol-3 ,1 1,20-trione.

V The reaction between the 16-methyl-4-pregnene-17a,21-diol-3,11,20-trione compound and formaldehyde to formthecorresponding bismethylenedioxy derivative is conveniently conducted byintimately mixing together a solution of the steroid in an organicsolvent, preferably a halogenated hydrocarbon solvent such aschloroform, and a solution of formaldehyde in an aqueous mineral acidsuch as aqueoushydrochloric acid. The reaction is ordinarily carried outat substantiallyroom temperature under which conditions the formation ofthe bismethylenedioxyderivative is substantially complete in about 2-3days. The non-aqueous layer is recovered, washed with 'anaqueous'alkaline' solutio'n'until neutral, dried and evaporated to givethe 17a,20,20,2l-bismethylenedioxy- 16nt-methyl-4-pregnene-3 ,1 l-dione.

The reaction between this bismethylenedioxy derivative and ethyleneglycol is carried out by heating the reactant's' together in solution ina hydrocarbon solvent such as benzene in the presence of an acidiccatalyst such as p-toluehesulfonic acid. Using benzene as the solvent,the reaction is conveniently conducted by heating the reaction solutionunder reflux for a period of about 15 hours, following which the cooledreaction solution is washed. with an aqueous alkaline solution, driedand evaporated to give 3-ethylenedioxy-17u,2O,20.21-bismethylenedioxy-16a-methyl-S-pregnene-1l-one. This 3-ethylenedioxy derivativecan be purified if desired by chromatography on acid washed alumina. V

The 3-ethylenedi0xy-17a,20,20,21-bismethylenedioxy-16ix-methyl-5-pregnene-1l-one is reacted with perbenzoic acid orperphthalic acid in the presence of a liquid medium, for example ahydrocarbon solvent such as benzene. The reaction is convenientlyconducted at room temperature under which conditions the reaction isordinarily substantially complete in about 2 days. The

reaction solution is decanted from a portion of theepoxide product, andthe remaining epoxide is recovered from the neutralized and driedreaction solution by evaporation. The epoxide product, that is the3-ethylenedioxy 17ot,20,20,21 bismethylenedioxy 5,6epoxylorx-methyl-pregnane-ll-one, is dissolved in substantiallyanhydrous formic acid, and the resulting solution allowed to stand atroom temperature for a period of about 2-3 hours. The reaction solutionis then poured into water and the aqueous mixture is extracted with ahalogenated hydrocarbon solvent such as; chloroform. The chloroformextract is neutralized, dried and evaporated to give a mixture of17a,20,20,21-bismethylenedioxy-l6amethyl-6-formyloxy-pregnane-5-ol-3,1l-dioneand 1704,20,

4 20,21 umwmylenedioxy" 16: methyl -5---tonny1oxypregnane-6-ol-3,11-dione. v

The reaction between the mixed formyloxy derivatives and the alkalihydroxide solution is conveniently conducted by dissolving the formyloxycompounds in a lower alkanol, adding to this solution an aqueoussolution of alkali metal hydroxide such as potassium hydroxide, andheating the resulting aqueous alcoholic alkaline solution under refluxandin the presence of a nitrogen atmosphere for aperiod of approximately/2 hour; The reaction solution is cooled, neutralized with an organicacid such as acetic acid, and evaporated to small volume. The aqueousconcentrate is diluted with water, and the aqueous mixture is extractedwith a halogenated hydrocarbon solvent such as chloroform. Thenon-aqueous extract is neutralized,- dried and evaporated to give 20,20, 2l-bismethylenedioxy-l6a-methylallopregnane-3,6,l l-trione, whichcan be purified, if desired, by crystallization from a lower alkanolsuch as methanol. a

The reaction between the17a,20,20,2I-bismethylenedioxy-16a-methyl-allopregnane-3,6,1l-trione andbutanone dioxolane is conveniently conducted by heating thereactants'together under reflux in the presence of an acidic catalystsuch as p-toluenesulfonic acid under which conditions the reaction isordinarily complete in about 10 minutes} The reaction mixture is cooled,diluted with chloroform and the resulting chloroform solution is washedwith bicarbonate, dried and evaporated to give 3 ethylencdioxy17a,20,20,21 bismethylenedioxy-l6mmethyl-allopregnane-6,ll-dione, whichcan be purified, if desired, by crystallization from ethyl acetate.

A solution of this 3ethylenedioxy-17a,20,20,2l-bismethylenedioxy-l6a-methyl-allopregnane-6,l1 dione in benzene is then added with stirring, over a 10 minuteperiod, to a solution of methyl magnesium iodide in diethyl ether. Theresulting. mixture is allowed to stir for about V2 hour additional time,water is added followed by additional benzene, and the organic layer isrecovered, dried and evaporated to give the corresponding3-ethylenedioxy-17a,20,20,21 bismethylenedioxy-6a,16t;-dimethylallopregnane-6fi ol-1l-one.

The reaction between the.3 ethylenedioxy-l7a,20,20,21-bismetl:tylenedioxy;- 60:,1611 dimethylallopregnane-Gflol-ll-one andthionyl chloride, when the latter is used as the dehydrating agent,is'conveniently conducted by adding. a solution of thionyl chloride inanhydrous pyridine drop-wise to an anhydrous pyridine solution of'thesteroid, while maintaining the temperature of the solution atapproximately 40 C. The resulting solution is stirred for an additional/2 hour period, cooled, poured into ice water, and the aqueous mixtureextracted with a halogenated hydrocarbon solvent such as chloroform. Thechloroform layer is neutralized, dried, and evaporated to a crudeproduce which is purified by chromatography over alumina to give3-ethylenedioxy-17u,20,20,21-bismethylenedioxy-6, 1 Ga-dimethyl-S-pregnene-1 l-one.

The. 3-ethylenedioxy 17a,2O,20,21 bismeth'ylenedioxy-6,16a-dimethyl-5-pregnene 1l-one is dissolved in anhydrous acetonecontaining p-toluenesulfonic acid monohydrate, and the resultingsolution is allowed to stand at room temperature for a period of about'15 hours. The resulting solution is then poured into water, theaqueous. mixture is extracted with a halogenated hydrocarbon; solventsuch as chloroform. The chloroform solution is neutralized,driedandevaporated to give 170:, 20,20,2labismethylenedioxy-6d,16adimethyl-4-pregnene- 3,11-dione. I, g

The reaction between the17u,20,20,2l-bismethylenedioxy-fia,16u-dimethyl-4-pregnene-3,1l-dioneand the aqueous organic acid hydrolyzing agent, more particula'rly. anaqueous acetic acid solution, is carried out by heating the reactantstogether at about 70 C. in a nitrogenatmosphere fora period of about 8hours. The hydrolysis solution is evaporated to give 6a,1'6a-dimethyl-4-pregnene-17a,21-diol-3,11,20-trione, which is then reacted with anacylating agent e,.g. acetic anhydride and pyridine to form thecorresponding 6a,16a-dimethyl-4- pregnene-17a,2l-diol-3,l1,20-trione2l-acylate.

The reaction between the hereinabove mentioned 3- ethylenedioxy17oz,20,20,21 bismethylenedioxy 6,160:- dimethyl-S-pregnene-l l-onecompound and lithium aluminum hydride is carried out by adding asolution of the steroid in a hydrocarbon solvent such as benzene to anethereal suspension of the lithium aluminum hydride and heating theresulting suspension under reflux for a period of about 4 hours. Ethylacetate is added to the cooled reaction mixture to destroy excesslithium aluminum hydride, water is then added, and the ethereal layer isrecovered, dried and evaporated to give a crude crystalline materialwhich is purified by chromatography over acid washed alumina to give3-ethylenedioxy-l7u,20,20, 2l-bismethylenedioxy-6,l6a-dimethyl-5-pregnene-l 1 5-01.

The reaction of the3-ethylenedioxy-l7a,20,20,21-bismethylenedioxy-6,l6a-dimethyl-5-pregnene-1lfi-oland ptoluenesulfonic monohydrate in acetone is conducted in the samemanner as described hereinabove in connection with the corresponding3-ethylenedioxy-l7a,20,20,21-bismethylenedioxy-6,16a-dimethyl-5-pregnene-1l-oneto produce 17a,20,20,2l-bismethylenedioxy-6a,l6a-dimethyl-4-pregnene-llfi-ol-Ii-one, which is then reacted with an aqueous organicacid hydrolyzing agent such as aqueous acetic acid in the same manner asthat set forth hereinabove in connection with the hydrolysis of the170:,20, 20,2l-bismethylenedioxy-6a,lfia-dimethyl 4 pregnene- 3,1l-dionecompound, thereby forming 6u,16a-dimethyl-4-pregnene-ll,8,17a,2l-trio1-3,20-dione, which is reacted with anacylating agent, e.g. acetic anhydride in pyridine to form thecorresponding 21-acylate such as60:,16a-dimethyl-4pregnene-l1B,17a,2l-triol-3,20-dione 2l-acetate, andthe like.

The reaction between the 6a,l6a-dimethyl-4-pregnene-11fl,l7a,2l-triol-3,20-dione 21-acylate and phosphorous oxychloride ormethane sulfonyl chloride is conducted by bringing the reactantstogether in pyridine solution at room temperature and allowing thesolution to stand at that temperature for a period of about hours. Thepyridine reaction solution is evaporated in vacuo to small volume,diluted with water and extracted with a waterimrniscible organic solventsuch as ethyl acetate. Evaporation of the washed and dried ethyl acetatesolution gives the corresponding 6m,l6a-dimethyl-4,9(ll)-pregnadiene-l7a,2l-diol 3,20-dione 2l-acylate which can be purified ifdesired by crystallization from an organic solvent such as ethylacetate-ether.

The addition of hypobromous acid to the M -double bond of this6a,16u-dimethyl-4,9(1l)-pregnadiene-l7a,21 diol-3,20-dione 21-acylate isconveniently carried out by adding an aqueous solution of perchloricacidto a suspension of the steroid compound and N-bromosuccinimide inaqueous dioxane while maintaining the temperature of the mixture belowabout 15 C. The reaction mixture is allowed to stand at 15 C. for about2-3 hours, the yellow solution is treated with allyl alcohol todischarge excess N-bromosuccinimide, and the resulting solution isevaporated in vacuo to small volume. The concentrated solution isdiluted with water and the aqueous mixture extracted with awaterimmiscible organic solvent such as ethyl acetate. The washed anddried ethyl acetate extract is evaporated to dryness to give thecorresponding 9a-bromo-6a,16a-dimethyl 4pregnene-llfi,l7a,2ltriol-3,20-dione Zl-acylate which can be purified,if desired by crystallization from an organic solvent such as ethylacetate-ether.

The reaction of this 9,1l-bromohydrin with potassium acetate is carriedout by heating the reactants together in ethanol under reflux for aperiod of about two hours. The cooled reaction mixture is evaporated tosmall volume in vacuo, the concentrated solution is diluted with water,and the aqueous mixture is extracted with ethyl acetate. The washed anddried ethyl acetate extract is evaporated in dryness in vacuo to givethe corresponding 6a,l6cz-dimethyl-9,ll-epoxy-4-pregnene-17a,21-diol-3,20-dione 21-acylate which is purified by crystallization from ethylacetate-ether.

The reaction between this 9,11-epoxide and hydrogen fluoride isconveniently conducted by bringing the reactants together in coldalcohol-free chloroform and allowing the resulting mixture to stand at 0C. for about two hours. A cold aqueous solution of sodium acetate isadded, and the resulting mixture is agitated vigorously. The layers areseparated, and the chloroform layer is washed, dried and evaporated todryness in vacuo to give 6a,].6oz-dimethYl-9oz-flll0l0 4pregnene-11/3,l7ot,21-triol- 3,20-dione 2l-acylate which can be purifiedby crystallization from acetone-petroleum ether.

This 6a,16a-dimethyl-9a-fluoro-4-pregnene-l1,8,17a,21- triol-3,20-dione2l-acylate is then dissolved in a mixture of benzene and dilutemethanolic potassium hydroxide and the mixture allowed to stand at roomtemperature for about 10 minutes. The reaction mixture is acidified withacetic acid, the solvents are evaporated in vacuo and the residualmaterial is crystallized from ethyl acetateether to give6a,l6u-dimethyl-9u fluoro-4-pregnene-llp, 17a,21-t1i01-3,20-di0116.

The oxidation of the llfi-hydroxyl group in the 604,160:-dimethyl-9a-fluoro 4 pregnene-l1,B,17a,2l-triol-3,20- dione 2l-acylateis conducted by dissolving the steroid ester in pyridine and adding thesolution to the complex formed by adding 1 part of chromium trioxide to5 parts of pyridine. The reactants are mixed thoroughly and the mixtureallowed to stand at room temperature about 15 hours. The reactionmixture is poured into water and the aqueous mixture is extracted withether and then with ethyl acetate. The combined organic extracts arewashed, dried and evaporated to dryness in vacuo to give6a,16a-dimethy1-9a-fluoro-4-pregnene-17 oc,21-diol-3,1 1,20- trione2l-acylate which can be purified by crystallization from ethylacetate-ether. This 2l-ester is hydrolyzed with methanolic potassiumhydroxide-benzene, in the same manner as described hereinabove inconnection with the hydrolysis of6a,16u-dimethyl-9u-fluoro-4-pregnene-1lfl, l7a,2l-triol-3,20-dione21-acylate, to produce60:,16a-dimethyl-9a-fiuoro-4-pregnene-17:1,21-diol-3,l 1,20-trione.

The microbiological A dehydrogenation of these 6:1,160: dimethyl 11oxygenated 4 pregnene 17oz, 2l-diol-3,20-dione compounds, e.g.6a,16ez-dim$thyi-4- pregnene 170:,21 diol 3,11,20 trione; 6u,16adimethyl 4 preguene 11B,17u,21 triol 3,26 dione; 6m,l6a dimethyl cfiuoro 4 pregnene 17a,21- diol 3,11,20 trione; 60;,160: dimethyl 9aiuoro 4- pregnene 115,17a,2l triol 3,20 dione; 21 esters of these60,16o: dimethyl 11 oxygenated 4 pregnenel7a,21-diol-3,20-dionecompounds, and the like, is conveniently conducted utilizingSchizomycetes microorganisms. This dehydrogenation reaction is efiectedby contacting the steroid compound with the Schizomycetes microorganismsthemselves or, if preferred, with enzyme systems of Schizomycetesmicroorganisms whereby the hydrogen attached to the *C-1 and C-2 carbonatoms is selectively removed to produce the corresponding A steroidsubstantially uncontaminated by unwanted products. When the6a,l6a-dimethyl-11-oxygenated-4-pregnene-l7oz,2l-diol-3,20-dionecompound is thus subjected to the dehydrogenating activity ofSchizomycetes microorganisms, the corresponding6u,16a-dimethyl-11-oxygenated 1,4 pregnadiene :,21 diol 3,20 dionecompound is obtained directly and in high yield. This microbiological Adehydrogenation procedure is ordinarily carried out by adding the6a,16a-din'1ethyl-ll-oxygenated 4 pregnene 170;,21 diol 3,20 dionecompound as a solid, or as a solution in a solvent as for example adialkyl ketone such as acetone, at dialkylformamide such asdimethyl-formamide, and the like, under sterile conditions to a cultureof the microorganism in a nutrient medium and agitating the resultingmixture thereby bringing about growth of the microorganism anddehydrogenation of the steroid compound.

7 The steroid can be added at the time the nutrient medium is inoculatedwith the culture of Schiz'omycetes' microorganisms or, alternatively,may be added to an established culture. Instead of adding the steroidcompound to the established culture in the nutrient medium, the cellgrowth from such established culture may be filtered from the broth,washed with distilled Water, then suspended in buffered aqueous solutioncontaining the 60 ,160; dimethyl 11 oxygenated 4 pregnene-1j7a,21-diol-3,20-dione compound, and the resulting mixutre agitatedthereby effecting dehydrogenation of the steroid compound to form thecorresponding 6a,16adimethyl 11 oxygenated 1,4 pregnadiene l7a,21-diol-3,20-dione. The latter is more readily recovered from this mediumthan from the mixture obtained when the steroid is incubated with themicroorganism in the original nutrient medium. Alternatively, the604,160;- dimet'nyl 11 oxygenated 4 pregnene 170:,21 diol- 3,20'-dionecompound may be contacted with dehydrogenating enzyme preparations fromthe growth of Schizo mycete's microorganisms.

The nutrient mediums used in carrying out this bacteriologicaldehydrogenation are those ordinarily utilized in the propagation ofSchizomycetes microorganisms. The usual nutrients include a nitrogen andcarbon source, inorganic salts and growth factors when required. Thecarbon can be provided by compounds such as acetates, lactates', and thelike. The nitrogen can be provided by an ammonium salt, amino acids, orproteins such as soy beans, oats, yeast, yeast extracts, tryptic digestof casein, meat extract, blood meal, protein meat and bone scrap, salmonmeal, fish meals, fish solubles, distillers solubles, and the like. Ifdesired, the Schizomycetes microorganisms can be propagated usingproteins (or amino acids) without any carbohydrate beingpresent in themedium, in which case the proteins or amino acids serve as the source ofboth the carbon and nitrogen required by the microorganisms,

While, as noted hereinabove, the dehydrogenation of the 60:,16d dimethyl11 oxygenated 4 pregnene- 17oz,2l'-diol-3,20-dione compound may becarried out using dehydrogenating enzyme preparations from the growth ofSchizomycetes microorganisms, or by contacting the steroid compound witha suspension of an established culture in distilled Water, it isordinarily preferred to add the 6a,l6ot-dirnethyl-ll-oxygenated-4-pregnone-170:1l-diol-3,20-dione compoundto a nutrient medium containing a 24-hour growth of Schizomycetesmicroorganisms. which may be added to the medium varies depending uponthe particular substrate being dehydrogenated, but it is ordinarilypreferred to employ a concentration of about 0.005% to 0.2% of6a,l6ocdimethyl 11 oxygenated 4 pregnene 170:,21 diol 3,20 dionecompound, although higher or lower concentrations may be employed, ifdesired. The culture containing the added steroid compound is thenincubated, preferably with agitation and aeration for an additionalperiod which ordinarily varies between about hours and 50 hours,although shorter or longer fermentation times may be advantageous forthe dehydrogenation of particular substrates. In view of the fact thatprolonged fermentations may result in destruction of a portion of the Adehydrogenated steroid product, it is ordinarily preferred to employ afermentation time of about 10 hours to 24 hours which, depending uponthe steroid substrate, has been found to result in maximal yields of theA dehydrogenated steroid product.

After completion of the dehydrogenation process, the 60a, 16a dimethyl11 oxygenated 1,4 pregnadiene-17a,2l-diol-3,20-dione product isconveniently recovered from the fermented broth by extraction with awater-immiscible solvent as for example a chlorinated hydrocarbon suchas chloroform, a ketone such as mehyl isobutyl keton'e, an alkylalkanoate such as ethyl ace-.

tate, and the like. The extract of A -dehydrogenated The proportion ofsteroid compound,

steroid product and any unreacted starting material which may be presentis conveniently purified by chroniatography using silica gel, activatedalumina, and the like or, if desired, by means of paper chromatograms.After separation of the dehydr'ogenated product from unreacted startingmaterial, the product can be purified further, if desired, byrecrystallization from a solvent.

such asethyl acetate, ethyl acetate-petroleum ether, and thelike.

in accordance with this microbiological dehydrogenation method, andusing the 6oz,l6a-dimethyl-ll-oxygenated-4-pregnene-l7a,21-diol-3,2Odione starting materials enumerated hereinbelow, there areobtained 6a.,dimethyl l1 oxygenated 1,4 pregnadiene l7u,2l-diol-3,20-dione compoundssuch as 6a,l6a-dimeth-' yl-l,4-pregnadiene-l7a,2l-diol-3,l1,20 trione;6a,l6a-dimethyl 1,4 pregnadiene 11,8,17a,21 triol 3,20 dione; 6a,16a-dimethyl-9cc-fiuoro-17a,2 l-diol-3 ,1 1,20-trione and6a,lfiwdimethyl-9ot-fiuoro-llB,l7a,2l-triol-3,20- dione.

Irrespective of whether the 6m,l6wdimethyl-1l-oxyg'enated-4-pregnene-1711,2.1-di0l-3,20-dione starting materialemployed in this microbiological dehydrogenation reaction is a 21-freealcohol or a 2l-ester thereof, the product obtained is the corresponding6a,16e-dimethyl- 11 oxygenated 1,4 pregnadiene l7oc,2l diol 3, 20-dione21-free alcohol, since any ZI-e'ster grouping which may be present ishydrolyzed during the micro-' diol3,20-dione 2l-frce alcohols can beconverted to the.

corresponding 2l-esters by reaction with an acylating agent e.g. aphosphorylating agent, a lower hydrocarbon carboxylic acid acylatingagent such as benzoic anhydride, tertiary butyl acetyl chloride, a loweralkanoic anhydride or lower al-kanoyl halide such as acetic anhydride,propionic anhydride, a polybasic acid anhydride such asfifi-dimethyl-glutaric anhydride, succinic anhydride, and the like.

In accordance with this acylation procedure there are obtained60:,16a-dimethyl l,4-pregnadiene-l7a,2ldiol-3,11,20-trione 2l-esters as,for example, 60:,16oc-dimethyl 1,4 pregnadiene :,21 diol 3,11,20 trioneZl-phosphate; 6a,lfiu-dimethyl-l,4-pregnadiene-l7a,2i-diol-3,ll,20-trione 2l 1ower hydrocarbon carbonyl esters such as6a,l6oz-dimethyl-1,4-pregnadiene-l7u,2ldiol 3,11,20 trione Zl-benzoate;6a,16a-dimethyl-l,4- pregnadiene-l7a,21-diol-3,l1,20-trione 2l-tertiarybutyl acetate; 6u,l6a dimethyl -l,4 pregnadiene-lhJl-diol- 3,11,20tri0ne I2l-lower alkanoates such as 6a,16a-dimethyl 1,4 pregnadiene17a,2l diol 3,11,20 trione 2 l-acetate; 60:, l 6m-dime'thyl-1,4-pregnadiene-1 70,2 l-diol- 3,11,20-trion 21-p-ropionate;6a,l6a-dil11ethyl-1,4-pregnadiene-l1,8,l7ot,2l-triol-3,20-dione 21-esters as, for example,6a,16a-dimethyl-1,4-pregnadiene-ll5,17u,2l-triol- 3,20-dione2l-phosphate; 6a,]Sa-dimethyl-1,4-pregnadiene llfi,l7a,21triol-3,20-dione 21-lower hydrocarbon carbonyl esters, such as6a,l6ot-dimethyl-l,4-pregnadiencllfl, 7a,21-triol-3,20-dioneZI-benzoate; 6a,l6a-dimethyl-l,4-pregnadiene-l1,3,17a,21-triol-3,20-dione 2l-tertiary butyl acetate;6e,l6a-dimethyl-1,4-pregnadiene-llfi,l7a, 2l-triol-3,20-dione 2l 'lowerallranoates such as 6a,16udirnethyl 1,4 pregn-adiene 11,8,170a,2l triol3,20 dione Zl-acetate; 6a,l6ot-dimethyl-l,4-pregnadiene-l118,l7a,2l-triol-3,20-dione 2l-prop-ionate; 9m-tluoro-6m,l6adimethyl 1,4pregnadiene l7u,2l diol-3,1130 trione 2l-esters as, for example,9oc-fluoro-6u,16a-dimethyl 1,4 pregnadiene-l7a,21-diol-3,l1,20-trionc2l-phosphate;9a-fluoro-6a,l6tx-dimethyl-l,4-prcgnadiene-l7e,2ldiol-3,ll,2Q-trione2l-lower hydrocarbon carbonyl esters such as 9ocfluoro-6a,l6u-dimethyl-1,4-pregnadiene-17a, 2 l-diol-3,l 1,20-tr-ione 2l-benzoate; 90c-fl1101'0-6rx,160c-dimethyl 1,4 pregnadiene 1701,21 diol3,11,20 trione 2l-tertiary butyl acetate; 9rx-flt1OI0-60c,16cx-dlmethyl-1,4-pregnadiene-l7a,21-diol-3,l1,20-trione 2l-lower alkanoates such as9-fluoro-6e,1fia-dimethyl-lA-pregnadiene-17e,21-diol-3,11,20-trione2l-acetate; 9a-fluo1'0-6u, 16cc dirnethyl 1,4 pregnadiene 1711,21 diol3,11, 20-trione 21-propionate; 9a-fiuoro-6a,16a-dimethyl-1,4-pregnadiene-l1B,17a,21-triol-3,20-dione 21 esters as, for example,9u-fiuoro-6a,16a-dimethyl-1,4-pregnadiene-1lfl, l7a,2l-triol-3,20-dione21-phosphate; 9ot-fll10I'O-6a,16ctdimethyl 1,4 pregnadiene 11,3,17a,21triol 3,20 dione 21-lower hydrocarbon carbonyl esters, such as 90:-fluoro 6:1,160: dimethyl 1,4 pregnadiene 1119,1741, 2l-triol-3,20-dione21-benzoate; 9a-fiu01'O-6cz,16a-dim6thyl 1,4 pregnadiene 1lp3,l7m,2ltriol 3,20 dione 21 -tertiary butyl acetate;9a-fiuoro-6a,l6a-dimethyl-1,4- pregnadiene 1le,17a,21 triol 3,20-dione2l-1ower alkanoates such as 9a-fiuoro-6a,IGa-dimethyI-IA-regnadiene-l15,17a,21-triol-3,20-dione 2l-acetate; 9a-fluoro-6a, 16mdimethyl 1,4 pregnadiene 1lfi,17a,21 triol 3,20-dione 21-propionate, andthe like.

Alternatively, instead of the above-mentioned microbiologicaldehydrogenation method, the 6a,l6a-dimethylll-oxygenated 4 pregnene- 17a,2 1-diol-3,20-dione compound is reacted with selenium dioxide therebyeffecting ring A dehydrogenation to form the corresponding 611,160:-dimethyl-l l-oxygenated-l,4-pregnadiene-17e,21-diol-3,20- dionecompound. This selenium dioxide dehydrogenation procedure isconveniently conducted by bringing the 61:,- lda-dimethyl-ll-oxygenated-4-pregnene-l7u,2l-diol- 3,20- dione compound, and seleniumdioxide together in the presence of an organic solvent such as forexample dioxane, an alcohol solvent such as t-butanol, etc., and heatingthe mixture at an elevated temperature. When t-butanol is used as thesolvent, it is ordinarily preferred to carry out this reaction at theboiling point of the solvent, under which conditions the reaction isordinarily complete in about fifteen hours. The reaction mixture isordinarily filtered, thereby removing metallic selenium, and thefiltered solution is evaporated to dryness in vacuo to give the desired6a,16a-dimethyl-11-oxygenated-l,4- pregnadiene-l7a,2l-diol-3,20-dionecompound. The crude material obtained in this way is convenientlypurified by paper strip chromatography in accordance with the procedureoutlined hereinabove in connection with the purification of the6a,l6a-dimethyl-l1-oxygenated-l,4-pregnadiene-17e,21-diol-3,20-dionecompound produced by microbiological dehydrogenation.

The following examples illustrate methods of carrying out the presentinvention but it is to be understood that these examples are given forpurposes of illustration an not of limitation.

Example 1 10 grams of l6a-methyl-4-pregnene-l7a,2l-diol-3,l1,20- trioneare dissolved in 400 cc. of chloroform. To this solution is added amixture of 100 cc. of concentrated aqueous hydrochloric acid and 100 cc.of 37% aqueous formaldehyde solution. The resulting mixture is stirredfor a period of approximately 3 days at room temperature. The chloroformlayer is separated, washed with sodium bicarbonate solution untilneutral, then with water, dried over anhydrous sodium sulfate, andevaporated to dryness in vacuo. The residual crude material is purifiedby crystallization from a mixture of methylene chloride and methanol togive l7a,20,20,2l-bismethylenedioxy-16a-methyl-4-pregnene-3 ,1 l-dione.

10 grams ofl7e,20,20,2l-bismethylenedioxy-l6a-methyl-4-pregnene-3,11-dione aredissolved in 500 cc. of henzene, to the solution is added 25 cc.ethylene glycol and 1 gram of p-toluenesulfonie acid, and the resultingmixture is heated under reflux for a period of approximately hours. Thereaction solution is cooled, washed with aqueous sodium bicarbonatesolution, dried over anhydrous sodium sulfate, and evaporated to drynessin vacuo. The residual crude material is adsorbed from its solution inbenzene on 250 g. of acid-washed alumina, and the resulting adsorbate iseluted utilizing mixtures of etherpetroleum ether to give3-ethylenedioxy-17a,20,20,21-bismethylenedioxy-16a-methyl-5-pregnene-1l-one.

A mixture of 10 grams of 3-ethylenedioxy-17a,20,20,21-bismethylenedioxy-l6a-methyl-5-pregnene-ll-one, 4.3 g. perbenzoic acidand 230 cc. benzene is allowed to stand at room temperature for a periodof about 2 days. A portion of the epoxide product, which precipitates,is separated from the reaction solution by decantation, and the decantedsolution is washed with saturated aqueous sodium bicarbonate solutionand then dried over anhydrous sodium sulfate. The dried solution is thenevap' orated in vacuo, and the residual epoxide product is combined withthe epoxide product originally separated from the reaction mixture.

The mixed epoxides are dissolved in 200 cc. of substantially anhydrousformic acid and the resulting solution is allowed to stand at roomtemperature for a period of about 2 /2 hours. The reaction solution ispoured into water, and the aqueous mixture is extracted with chloroform.The chloroform solution is washed with saturated aqueous sodiumbicarbonate solution, dried over anhydrous sodium sulfate, andevaporated in vacuo to give a mixture of17a,20,20,2l-bismethylenedioxy-lGamethyl-6-formyloxy-pregnane-5-ol-3,ll-dioneand 1701,20,- 20,21-bismethylenedioxy-l6u-methyl-5 formyloxypregnane-6-ol-3.11-dione.

This mixture of S-formyloxy and 6-formyloxy derivatives is dissolved inabout 850 cc. of methanol, to this solution is added a solutioncontaining about 17 grams of potassium hydroxide in cc. of water, andthe resulting mixture is heated under reflux in a nitrogen atmospherefor a period of approximately V2. hour. The reaction solution is cooled,neutralized with about 23 cc. of acetic acid and evaporated in vacuo toa small volume. The concentrated solution is poured into water and theaqueous mixture is extracted with chloroform. The chloroform extract isWashed with aqueous sodium bicarbonate solution, dried over anhydroussodium sulfate, and evaporated to dryness in vacuo. The residualmaterial is crystallized from methanol to give17a,20,20,21-bismethylenedioxy-l6a-methyl-allopregnane-3 ,6,l l-trione.

A solution containing about 5 grams of 17a,20,20,21- bismethylenedioxy16o: methyl-allopregnene-3,6,1l-trione and 0.1 g. of p-toluenesulfonicacid in cc. butanone dioxolane is heated under reflux for a period ofabout 10 minutes. The reaction solution is cooled to about 0-5 C.,diluted with chloroform, and the resulting solution is Washed withaqueous sodium bicarbonate solution and dried over anhydrous sodiumsulfate. The resulting dried solution is evaporated to dryness in vacuo,and the residual material is slurried with ether, recovered byfiltration and then dried to give 3-ethylenedioxy-l7a, 20,20,21bismethylenedioxy-l6a-methyl-allopregnane-6, ll-dione.

A solution containing about 5 grams of 3-ethylenedioxy17a,20,20,2l-bismethylenedioxy-l6a-methyl-allopregnane-6,11-dione in 70cc. of benzene is added, with stirring over a period of approximately 10minutes, to an ethereal solution containing the methyl magnesium iodide,prepared from 5 cc. of methyl iodide and 0.5 gram of magnesium turnings,dissolved in 50 cc. of ether. The reaction mixture is allowed to stirfor an additional /2 hour period, and the resulting solution isdecomposed with 7 0 cc. of water. About cc. of benzene is added to theaqueous mixture and the layers are separated. The aqueous layer isextracted with two 100 cc.-portions of chloroform, and the organiclayers are combined, dried over anhydrous sodium sulfate, and evaporatedto dryness in vacuo to giveS-ethylenedioxy-17a,20,20,2l-bismethylenedioxy6a,16e-dimethyl-allopregnane-6 3-01-11- one.

A solution containing 5.5 cc. of freshly distilled thionyl chloride in26 cc. of cold anhydrous pyridine is added dropwise, with stirring, to asolution of 5.0 g. of 3-ethylenedioxy 17a,20,20,21 bismethylenedioxy-6o1Got-di- I methyl-allopregnene-GB-ol-lLone in 32 cc of anhydrouspyridine, while maintaining the temperature of the reaction mixture atapproximately 40 C. The reaction solution is stirred for an additional30 minute period following the addition of the thionyl chloride reagent,and the reaction mixture is then cooled to about 5 C. and poured into180 cc. icewater. The aqueous mixture is' extracted with chloroform,andthe chloroform extract is neutralized, dried over anhydrous sodiumsulfate, and evaporated to dryness in vacuo. The residual material,dissolved in benzene, is chromatographed on 150 grams of acidwashedalumina; elution of the resulting adsorbate with mixtures of petroleumether and ether gives 3-ethylenedioxy17a,20,20,21-bismethylenedioxy-6,16udimethyl-5- pregnene-ll-one. a

A solution containing about 5 grams of 3-ethylenedioxy 17a,20,20,21bismethylenedioxy-6,16a-dimethy1-5- pregnene-ll-one, 500 cc. ofanhydrous acetone and about 0.5 g. of p-toluenesulfonic acid monohydrateis allowed to stand for a period of about hours. The reaction solutionis poured into water, and the aqueous mixture is extracted withchloroform. The chloroform solution is washed with aqueous sodiumbicarbonate solution, dried over anhydrous sodium sulfate, and thechloroform solution is evaporatedto dryness in vacuo. The residualmater'ial is crystallized from benzene to give 17a,20,20,21-bismethylenedioxy 6a,16u dimethyl- 4-pregnene-3,1ldione.

About 0.1 gram of 17a,20,20,2l-bismethylenedioxy-Ga,16ot-dimethyl-4-pregnene3,ll-dione is suspended in 18 cc. of 50% aqueousacetic acid, and the suspension is carefully purged with nitrogen,following which the suspension isheated on a steam bath under nitrogenfor a period of about 8 hours and then allowed to stand at roomtemperature for an additional period of 15 hours. The reaction solutionis evaporated to dryness in vacuo to give 601,160:dimethyl-4-pregnene-l7u,21-diol-3,11,20-trione. This material isdissolved in 20 cc. of chloroform, and the chloroform solutionevaporated to dryness in vacuo. To the residual dry material is added 1cc. of pyridine and 1 cc. ofacetic anhydride, and the resulting mixtureis heated on a steam bath for a period of about 15 minutes. The reactionsolution is cooled to about (i-5 C. and poured into ice water. Theaqueous suspension is extracted with chloroform, and the chloroformextract is washed with aqueous sodium bicarbonate solution, 7 a driedover anhydrous sodium sulfate, and'evaporated to ether-chloroform, andthen with 50 cc. of a 3:7 mixture of ether-chloroform. The eluates areevaporated in vacuo, and the residual material is crystallized frommethanol to give 6a,l6a-dimethyl-4 pregnene-17a,21-diol-3,l1, ZO-trioneZI-acetate.

To a solution of 100 mg. of6u,16a-dimethyl-4-pregnene-17a,21-diol-3,l1,20-trione 21-acetate, in 6ml. tbutanol, and 0.01 ml. of glacial acetic acid is added 70 mg. ofselenium dioxide. The mixture is heated under reflux in a nitrogenatmosphere for a period of about 6 hours. The reaction solution isfiltered and evaporated Example To a solution of 100 mg. of 17 u,20,20,2 l-bismethylene- 7 diox -6a,l6a-dimethyl-4-pregnene-3, l1-dione,prepared as describedin Example 1 hereinabove, in 6 m1. t-butanol, and0.01 ml. of glacial acetic acid is added 70 mg. of selenium dioxide. Themixture is heated under reflux ina nitrogen atmosphere for a period ofabout 6 hours. The reaction solution is filtered and evaporated invacuo. The residual material is dissolved in benzene, and the benzenesolution is Washed with an aqueous sodium bicarbonate solution and driedover anhydrous sodium sulfate. Thebenzene solution is stirred withmercury overnight, then centrifuged, and the clear benzene solution isevaporated in vacuo. The residual material thus obtained isseparatedusing partition chromatography on diatomaceous silica(supercel) to give 17a,20,20,21-bi5- methylenedioxy 60:,160: dimethyl1,4 pregnadiene- 3,11-dione.

About 0.1 gram of -17a,20,20,2l-bismethylenedioxy-fia,l6u-dimethyl-1,4-pregnadiene-3,ll-dione is suspended in 18 cc. of 30%aqueous acetic acid, and the suspension is carefully purged withnitrogen, following which the suspension is heated on. a steam bathunder nitrogen for a period of about 8 hours and then allowed to standat room temperature for an additional period of 15 hours. The reactionsolution is evaporated to dryness in vacuo to give6a,lou-dimethyl-1,4-pregnadiene-17a,21-cliol-3,ll, ZO-trione. Thismaterial is dissolved in 20 cc. of chloroform, and the chloroformsolution evaporated to dryness in vacuo. To the residual dry material isadded 1 cc. of pyridine and 1 cc. of acetic anhydride, and the resultingmixture is heated on a steam bath for a period of about 15 minutes. Thereaction solution is cooled to about 0-5" C. and poured into ice water.The aqueous suspension is extracted with chloroform, and the chloroformextract is washed with aqueous sodium bicarbonate solution, dried overanhydrous sodium sulfate, and evaporated to dryness in vacuo. Theresidual material, dissolved in 30 cc. benzene, is adsorbed on 5 gramsof acid- Washed alumina; the adsorbate is washed with 50 cc. ether. Theadsorbate is then eluted with 250 cc. of a 5:5 mixture ofether-chloroform, and then with 50 cc. of a 3:7 mixture ofether-chloroform. The eluates are evaporated in vacuo, and the residualmaterial is crystallized from methanol to give6u,1Ga-dimethyl-I,4-pregnadienel7a,2l-diol-3, 1 1,20-trione 21-acetate.

Example 3 About 5 grams of3-ethylenedioxy-17a,20,20,2l-bismethylenedioxy 6,1611 dimethyl 5pregnene l1 one, which can be prepared as described in Example 1hereinabove, is dissolved in about cc. of benzene. This solution isadded to a stirred suspension containing 5 grams of lithium aluminumhydride in one liter of anhydrous ether, and the resulting suspension isheated under refluxfor a period of about 4 hours. The reaction mixtureis allowed to cool to about room temperature, and approximately 33 cc.of ethyl acetate is added to destroy excess lithium aluminum hydride.The resulting mixture is admixed with about cc. of water, the ethereallayer is decanted, and the aqueous layer, diluted with an additional 200cc. of water, is extracted with chloroform. The ether and chloroformlayers are com bined, and the combined organic layers are dried overanhydrous sodium sulfate and evaporated to dryness in vacuo. Theresidual material, dissolved in about 250 cc. of benzene, is adsorbed onabout grams of acidwashed alumina, and the resulting adsorbate is elutedutilizing mixtures of petroleum ether-ether. This eluate is evaporatedin vacuo, and the residual material is purified by crystallization fromether to give 3-ethylenedioxyl7u,20,20,2l bismethylenedioxy 6,160:dimethyl 5- pregnene-l lfi-ol.

A solution containing about 5 grams of 3-ethylenedioxy :,2O,20,21bismethylenedioxy 6,160; dimethyl-S-pregnene-llfl-ol, 500 cc. ofanhydrous acetone and about 0.5 g. of p-toluenesulfonic acid monohydrateis allowed tostand for a period of about 15 hours. The

reaction solution is poured into water, and the aqueous mixture isextracted with chloroform. The chloroform solution is washed withaqueous sodium bicarbonate solution, dried over anhydrous sodiumsulfate, and the chloroform solution is evaporated to dryness in vacuo.The residual material is crystallized from benzene to give 17a,20,20,21bismethylenedioxy 6a,16a. dimethyl 4- pregnene-l lB-ol-3-one.

About 0.1 gram of l7a,20,20,2l-bismethylenedioxy-6e,l6oc-dimethyl-4-pregnene-l1 B-ol-3-one is suspended in 18 cc. of 50%aqueous acetic acid, and the suspension is carefully purged withnitrogen, following which the suspension is heated on a steam bath undernitrogen for a period of about 8 hours and then allowed to stand at roomtemperature for an additional period of 15 hours. The reaction solutionis evaporated to dryness in vacuo to give6a,16u-dimethyl-4-pregnene-1lfi,l7a,21-triol-3,20- dione. This, materialis dissolved in 20 cc. of chloroform, and the chloroform solutionevaporated to dryness in vacuo. To the residual dry material is added 1cc. of pyridine and 1 cc. of acetic anhydride, and the resulting mixtureis heated on a steam bath for a period of about 15 minutes. The reactionsolution is cooled to about -5 C. and poured into ice water. The aqueoussuspension is extracted with chloroform, and the chloroform extract iswashed with aqueous sodium bicarbonate solution, dried over anhydroussodium sulfate, and evaporated to dryness in vacuo. The residualmaterial, dissolved in 30 cc. benzene, is adsorbed on grams ofacid-washed alumina; the adsorbate is Washed with 50 cc. ether. Theadsorbate is then eluted with 250 cc. of a 5:5 mixture or"ether-chloroform, and then with 5 0 cc. of a 3:7 mixture ofether-chloroform. The eluates are evaporated in vacuo, and the residualmaterial is crystallized from methanol to give 60:,16oc-dlH1ETlIYl-4-pregnene-l1 3,17a,2l-triol-3,20-dione 2l-acetate.

To a solution of 100 mg. of6a,16a-dimethyl-4-pregnene-l1,B,17e,21-uiol-3,20-dione 2l-acetate, in 6m1. t-butanol, and 0.01 ml. of glacial acetic acid, is added 70 mg. ofselenium dioxide. The mixture is heated under reflux in a nitrogenatmosphere for a period of about 6 hours. The reaction solution isfiltered and evaporated in vacuo. The residual material is dissolved inbenzene, and the benzene solution is washed with an aqueous sodiumbicarbonate solution and dried over anhydrous sodium sulfate. Thebenzene solution is stir-red with mercury over night then centrifugedand the clear benzene solution is evaporated in vacuo. The residualmaterial thus obtained is separated using partition chromatography ondiatomaceous silica (supercel) to give6a,l6u-dimethyl-1,4-pregnadiene-l1fi,l7a,2l-triol-3,20-dioneill-acetate.

Example 4 To a solution of 100 mg. of17a,20,20,2l-bismethylenedioxy-6a,l6e-dimethyl-4-pregnene-11,3-ol-3-one,prepared as described in Example 1 hereinabove, in 6 ml. t-butanol, and0.01 ml. of glacial acetic acid is added 70 mg. of selenium dioxide. Themixture is heated under reflux in a nitrogen atmosphere for a period ofabout 6 hours. The reaction solution is filtered and evaporated invacuo. The residual material is dissolved in benzene, and the benzenesolution is washed with an aqueous sodium bicarbonate solution and driedover anhydrous sodium sulfate. The benzene solution is stirred withmercury overnight then centrifuged and the clear benzene solution isevaporated in vacuo. The residual material thus obtained is separatedusing partition chromatography on diatomaceous silica (supercel) to givel7u,20,20,2l-bismethylenedioxy-6a, l oa-dirnethyl-1,4-pregnadiene-1 lfl-ol-S-one,

About 0.1 gram of 17a,20,20,2l-blSInEthYlCHBdiOXY-Sa,la-dimethyl-l,4-pregnadienc-llB-ol-3-one is suspended in 18 cc. of 50%aqueous acetic acid, and the suspension is carefully purged withnitrogen, following which the suspension is heated on a steam bath undernitrogen for a period of about 8 hours and then allowed to stand at roomtemperature for an additional period of 15 hours. The reaction solutionis evaporated to dryness in vacuo to give6a,la-dimethyl-1,4-pregnadiene-1l5,l7a,21-triol- 3,20-dione. Thismaterial is dissolved in 20 cc. of chloroform, and the chloroformsolution evaporated to dryness in vacuo. To the residual dry material isadded 1 cc. of pyridine and 1 cc. of acetic anhydride, and the resultingmixture is heated on a steam bath for a period of about 15 minutes. Thereaction solution is cooled to about 05 C. and poured into ice water.The aqueous suspension is extracted with chloroform, and the chloroformextract is washed with aqueous sodium bicarbonate solution, dried overanhydrous sodium sulfate, and evaporated to dryness in vacuo. Theresidual material, dissolved in 30 cc. benzene, is adsorbed on 5 gramsof acid-washed alumina; the adsorbate is washed with 50 cc. ether. Theadsorbate is then eluted with 250 cc. of a 5:5 mixture ofether-chloroform, and then with 50 cc. of a 3:7 mixture ofether-chloroform. The eluates are evaporated in vacuo, and the residualmaterial is crystallized from methanol to give6a,l6a-dimethyl-l,4-pregnadiene-l113, 17e,2l-triol-3,20-dione2l-acetate.

Example 5 To a cooled solution of 436 mg. of 6m,l6a-dimethyl-4-pregnene-l118,17a,21-triol-3,20-dione 21-acetate (which can be preparedas described in Example 3 hereinabove) in 2.5 cc. dimethyl formamide and2.0 ml. of dry pyridine is added 1.0 ml. of methane sulfonyl chloride,While maintaining the temperature below 0 C. The resulting mixture isallowed to warm to room temperature, at which point a precipitateappears; the resulting mixture is then heated to a temperature of about70-100 C. for a period of about 10 minutes. About 15 ml. of water isadded slowly to the reaction mixture, with stirring, and the aqueousmixture is extracted with ethyl acetate. The combined ethyl acetateextracts are washed with water, then with dilute aqueous hydrochloricacid solution, again with water, and then with a dilute aqueous sodiumbicarbonate solution. The washed ethyl acetate solution is dried, andthe solvent is evaporated in vacuo; the residual material is trituratedwith ether, and the crystalline material is recrystallized to give6u,l6ot-dimethyl-4,9(1l)-pre-gnadiene-l7a,2l-diol-3,20-dione Zl-acetate.A suspension of 330 mg. of 6a,l6a-dimethyl-4,9(ll)-pregnadiene-17a,21-diol-3,20-dione 21-acetate and 1.8 g. of N-bromo-succinimide in amixture of 50 ml. of dioxane and 10 ml. of water is cooled to 10 C. Thenwith stirring, 10 ml. of cold 1.0 N aqueous perchloric acid is added.The temperature of the reaction mixture is allowed to rise to 15 C. andis maintained at this point for about two and onehalf hours during whichtime the solid material slowly dissolves. The resulting yellow solutionis treated with 1.0 ml. of allyl alcohol to discharge the color and theremaining N-bromo-succinimide, and the resulting solution is evaporatedto a small volume in vacuo. The concentrated solution is diluted withwater, and the aqueous mixture is extracted with ethyl acetate. Theethyl acetate extracts are washed, dried and evaporated to dryness, andthe residual material is crystallized from ethyl acetateether to give9a-bromo-6a,l6a-dimethyl-4-pregnene-l1B, l7u,2l-triol-3,20-dioneZl-acetate.

A solution of 210 mg. of 9a-bromo-6a,l6a-dimethyl-4-pregnene-llB,l7a,2l-triol-3,20-dione 2l-acetate and 240 mg. of potassiumacetate in 10 ml. of absolute ethanol is heated under reflux for twohours. The reaction mixture is cooled to room temperature, evaporated invacuo to a small volume, and diluted with water. The concentratedaqueous mixture is extracted with three portions of ethyl acetate, andthe combined ethyl acetate extracts are washed with water, dried, andevaporated to dryness in vacuo. The residual material is crystallizedfrom ethyl acetate-ether to give6a,16a-dimethy1-9,ll-epoxy-4-pregnene-17a,2l-diol-3,20-dione Zl-acetate.

To a solution of 83 mg. of anhydrous hydrogen fluoride in 4.7 'ml.'ofice cold alcohol-free chloroform is addedsolution is mixed thoroughlyand maintained at C.

for two hours, at the end of which time 13.0 ml. of icecold 20% aqueoussodium acetate is added, and the resulting mixture agitated vigorously.The layers are sepa rated, and the chloroform layer is washed with wateruntil free of acid, dried, and the chloroform evaporated in vacuo.' Theresidual material is crystallized from acetone-petroleum ether to give6a,16a-dimethyl-9afluoro-4-pregnene-115,17a,21-triol-3,20-dione21-acetate. Fifty milligrams of 6a,16a-dimethyl-9m-fluoro-4-pregnene-11,8,17a,2l-t1i01-3,20-dl0116 21-acetate is dissolved in a mixture of1.0 cc. of benzene and 1.0 cc. of 1 N methanolic potassium hydroxide,and the solution is allowed to stand at room temperature for a period ofabout minutes. The solution is then acidified with acetic acid, thebenzene is evaporated in vacuo, and the residual material is purified bycrystallization to give 6a,16c-dimethyl- 9a-fi-uoro-4-pregnene-l113,17a,21-t1i0l-3,20-dl01l6.

To a solution of 100 mg. of16a,16a-dimethyl-9afluoro-4-pregnene-l1B,l7u,21-triol-3,20-dione21-acetate, in 6 ml. t-butanol, and 0.01 ml. of glacial acetic acid isadded 70 mg. of selenium dioxide. The mixture is heated under reflux ina nitrogen atmosphere for a period of about 60 hours. The reactionsolution is filtered and evaporated in vacuo. The residual material isdissolved in benzene, and the benzene solution is washed with an aqueoussodium bicarbonate solution and dried over anhydrous sodium sulfate. Thebenzene solution is stirred with mercury overnight, then centrifuged,and the clear benzene solution is evaporated in vacuo. The residualmaterial thus obtained is separated, using partition chromatography ondiatomaceous silica (supercel), to give 60c,16wdimethyl-9a-fiuoro-1,4-pregnadiene-11;3,17a,21 triol- 3,20-dione21-acetate. This material is hydrolyzed using 1 N methanolic potassiumhydroxide in benzene as described above for the hydrolysis of6a,16u-dimethyl-9afluoro-4-preguene-11p,17a,21-triol-3,20 dioneZI-acetate 17a,2l-triol-3,20-dione.

Example 6 A solution of 400 mg. of 6a,16a-dimethyl9a-fiuoro--4-pregnene-l15,l7a,2l-triol-3,20-dione 21-acetate in 4 ml. of pyridineis added to the complex formed by the addition of 400 mg. of chromiumtrioxide to 4 ml. of pyridine. The mixture is swirled until thoroughlymixed and then allowed to stand at room temperature overnight. Thereaction mixture is poured into water, and the aqueous mixture isextracted with ether and then twice with ethyl acetate. The combinedether and ethyl acetate extracts are washed with dilute aqueous sulfuricacid at about 0 C., and then with water until neutral. The organicsolvent layer is then dried, the solvents are evaporated therefrom invacuo, and the residual crystalline material is purified bycrystallization to give6d,l6u-dimethyl-9otfiuoro-4-pregnene-17a,21-diol-3,11,20-trione 21acetate. Fifty milligrams of 61x,16a-dimethyl-9a-fiuoro-4-pregnene-170;,21-di0l-3,11,2O-trione 2l-acetate is dissolved in a mixture of 1.0cc. of benzene and 1.0 cc. of l N methanolic potassium hydroxide, andthe solution is allowed to stand at room temperature for a period ofabout 10 minutes. The solution is then acidified with acetic acid, thebenzene is evaporated, in vacuo, and the residual material is purifiedby crystallization to give 6u,l6a-dimethyl-9a-fluoro-4-pregnene-17a,2l-diol-3,11,20-trione.

To a solution of 100 mg. of 6a,16u-dimethyl 9u-fluoro-4-pregnene-l7a,2l-diol-3,20-trione 21-acetate, in 6 ml. tbutanol, and0.01 ml. of glacial acetic acid is added 70 mg. of selenium dioxide. Themixture 'is heated under reflux in a nitrogen atmosphere for a period ofabout 60 hours. The reaction solution is filtered and evaporated invacuo. The residual material is dissolved in benzene,

Example 7 Fifty milliliters of a nutrient medium are prepared having thefollowing composition:

Cerelose g 1 Edamin a 1 Cornsteep liquor ml 0.25

Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculatedwith about 2.5 to 5 ml. of a culture of Bacillus spha'ericus (ATCC-245)microorganisms, and the inoculated culture is then incubated at atemperature of 28 C., with agitation, for a 24-hour period. To theresulting culture is added a solution containing 10 mg. of6a,16a-dimethy1-4-pregnene-l7a,2l-diol-3,l1,20- trione dissolved in 0.2nil. of dimethylformamide. The culture containing the steroid compoundis. incubated, with agitation, for an additional period of about 24hours at 28 C.

The fermentation broth is extracted with four 50' ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporatedinvacuo to a volume of about 5 ml. The concentrated solution is thenstreaked on paper chromatograms which are developed using formamide asthe stationary phase and 50% benzene-50% chloroform as the mobile phase.After 8 hours development in a descending system, the upper bands foreach chromatogram, corresponding to the A -dehydro derivative, are cutoff, extracted with methanol, and the methanol-extracted material isagain subjected to streak-paper chromatography. The upper band is againcut oil, thoroughly dried, extracted with methanol, and the methanolextract is evaporated to dryness in vacuo. The residual material isrecrystallized to give 6a,16a-dimethyl-1,4-pregnadiene-17u,2l-diol-3,l1,20-trione.

The 60,l6a dimethyl 1,4-pregnadiene-17a,21-dio1- 3,11,20-trioneistreated with aceticv anhydride and pyri dine to give the 2l-acetylderivative, which is purified by recrystallization to give substantiallypure 60:,l6u-dimethyl 1,4-pregnadiene-l-7u,21-diol-3,11,20-trione 21acetate.

Example 8 I Fifty milliliters of a nutrient medium are prepared havingthe following composition:

Ccrelose E 1 Edamin g 1 Cornsteep liquor ml 0.25

Distilled water to make 50 ml.

portions of ethyl acetate, and the ethyl acetate extracts are combinedand evaporated in vacuo to a volume of about ml. The concentratedsolution is then streaked on paper chromatograms which are developedusing formamide as the stationary phase and 50% benzene- 50% chloroformas the mobile phase. The upper bands for each chromatogram correspondingto the A -dehydro derivative are cut off, extracted with methanol, andthe methanol-extracted material is again subjected to streakpaperchromatography. The upper band is again cut 01?, thoroughly dried,extracted with methanol, and the methanol extract is evaporated todryness in vacuo. The residual material is recrystallized to give6a,16u-dimethyl- 1,4-pregnadiene-l1B,l7a,21-triol-3,20-dione.

The 6a,lfiu-dimethyl-1,4-pregnadiene-l1,8,17a,21-triol- 3,20-dione istreated with acetic anhydride and pyridine, and the acetylated productrecrystallized to give substantially pure6a,l6a-dimethyl-1,4-pregnadiene-11fi,17a,21- triol-3,20-dione21-acetatc.

Example 9 This medium is adjusted to pH 6.5 with KOH, sterilized andinoculated with about 2.5 to 5 ml. of a culture of Mycobacteriumsmegmatis (NRRL B-1667) micro organisms, and the inoculated culture isthen incubated at streak-paper chromatography, using paper which hasbeen extracted for 48 hours with methanol, and employing thechloroform-formamide system previously employed. The resultingchromatogram shows only a trace band corresponding to the startingmaterial with the majorband that corresponding to the less mobilecomponent, the A -dehydro derivative. The paper chromatogram isthoroughly dried, and the band corresponding to the less mobilecomponent is cut ofli and extracted with methanol. The methanol extractis evaporated to dryness in vacuo to give 6a,16u-dimethyl-9fluoro1,4-pregnadiene-1lfi,17a,2l-t1iol-3,20-dione.

The 601,161: dimethyl 9oz fiuoro 1,4pregnadienel1B,17a,2l-triol-3,20-dione is reacted with excess aceticanhydride in pyridine to give the 21-acetyl derivative which is purifiedby recrystallization from benzene-petroleum ether to give substantiallypure 6a,16a-dimethyl- 9a fluoro 1,4 pregnadiene 1l13,l7a,2l triol 3,20-dione 21-acetate.

Example 10 The fermentation procedures of Examples 7, 8 and 9 arerepeated but using, in place of the microorganisms and the6a,la-dimethyl-ll-oxygenated-4-pregnene-l7a, 2l-diol-3,20-dione startingmaterial employed in those examples, the microorganism strains andsteroid starting compounds indicated in the table hereinbelow. Theresulting fermentation broths are treated in accordance with theisolation methods described in Examples 7, 8 and 9 to give, for theparticular microorganism strain and steroid substrate used, the6a,l6u-dimethyl-ll-oxygenated-l,4-pregnadiene-l7a,2l-diol-3,20-dioneindicated in the following table:

Expt. 6a,16a-D1methyl-11-oxygen- N0. Substrate Microorganismated-1,4-Pregnadiene-17a, 21-Dlol-3,20-Dlone Product 1 601,160:Dirnethyl 90: flu- .Ba illus sphaericus Se led Dlmethyl 9c: fluoro 4pregnene 170 21 ATOC-7055 oro 1,4 pregnadiene 17a, diol-3,11,20trione21-ace- 21-diol-3,11,20-trione.

ate.

2 6:2,162 Dimethyl 9a fiu- Bacillus sphaerz'cus 611.1%: Dimethyl 9afluoro 4 pregnene 116,1701, ATTC-7063. cro 1,4 pregnadiene 11B, 21 triol3,20 dione 21- 17a,21-trlol-3,20-dione. acetate.

3 611,161: Dimethyl 9e: fiu- Nocardia Zeish- 6a.16a Dimethyl 9a fluoro 4preguene 17,21- manii AICC- oro 1.4 pregnadiene 17a,diol-3,11,20-trione. 6855. 2l-diol-3,11,20-trione.

4 60:,16a Dimethyl 9oz flu- Nocardia formica 60,1601 Dimethyl 9a fluoro4 pregnene 118,17a, NRRL-2470 oro 1,4 pregnadiene 11B,21-triol-3.20-dione. 17a 21-triol-3,20dione.

5 5:2,16a Dirnethyl 4 preg- Mycobacierium 641,16: Dimethyl 1,4 pregnene11B,17a,2l triol 3, phlei ATCO- nadiene 11B,17a,21 triol- 20-dlone.12,298. 3,20-dione.

6 601,16: Dimethyl 4 preg- Mycabacterium 611,160; Dimethyl 1,4 pregnene1711,21 diol 3,11,20- lactic-Ola ATOC- nadiene 170;,21 diol 3,11,trione. 12,297. ZO-trione.

a temperature of 28 C., with agitation, for a 24-hour period. To theresulting culture is added a solution containing 10 mg. of6a,16a-dimethyI-Qa-fluoro-4-pregnene- 11[3,17a,21-t1i013,20-dl0118dissolved in 0.2 ml. of dimethylformamide. The culture containing thesteroid compound is incubated, with agitation, for an additional periodof about 24 hours at 28 C.

The fermentation broth is extracted with three ml.- portions of ethylacetate, and the ethyl acetate extracts are combined and evaporated invacuo to a volume of about 5 ml. The concentrated solution is then usedto prepare streak-paper chromatograms which are developed utilizingformamide as the stationary liquid phase and 50% benzene-50% chloroformas the mobile liquid phase. Two bands are secured, one of whichcorresponds to the more mobile component, the 6a,l6a-dimethyl 9a fluoro4 pregnene 1lfi,l7oc,2l triol- 3,20- dione starting material, and theother corresponds to the less mobile component, the A -dehydroderivative. The paper chromatogram is dried, and the latter band is cutoff and extracted with methanol. The material extracted with methanol isagain subjected to The 16 methyl 4 pregnene :,21 diol 3,11,20-. trionecompound used as starting material in the foregoing examples isprepared, starting with the known 16- pregnene-3a-ol-11,20-dione3-acetate, in accordance with the following procedure:

A solution of 10.22 g. of methyl iodide in 50 ml. of ether is added to1.73 g. of magnesium in 50 ml. of ether. To the resulting etherealsolution of methyl magnesium iodide, maintained under a nitrogenatmosphere, is added 0.045 g. of anhydrous cuprous chloride. To thismixture is added, over a period of about one hour, during which periodthe reaction mixture is stirred vigorously and maintained atapproximately room temperature, a solution of about 5.6 g. of 16-pregnene-3a-ol-1l, ZO-dione 3-acetate in ml. of ether. A white granularsolid separates during this addition. The resulting mixture is heatedunder gentle reflux for two hours after which the reaction mixture iscooled, and 125 ml. of saturated, aqueous ammonium chloride solution isadded followed by 200 ml. of ether. The layers are separated, and theethereal layer is washed with three 50 ml. portions of water. The washedethereal layer is dried, and

' With Z liters of petroleum ether.

the solvent evaporated in vacuo to give a brown viscous oil. The lattermaterial is heated for 15 minutes at 60-70 C. with a mixture of 25 ml.acetic anhydride and 25 ml. pyridine and the acetylated product ispurified by chromatography on acid-washed alumina followed bycrystallization from petroleum ether to give approximately 1.5 g; ofsubstantially pure l6a-methyl-pregnene- 3d-ol-ll,20-dione 3-acetate.

To a solution of 0.8 g. of l6u-methyl-pregnane-3a ol- 1.1,20-dione3-acetate in 40 ml. of methanol is added 1.5 ml. of concentrated aqueoushydrochloric acid and the resulting solution is stirred overnight atabout 25 C. The reaction solution is evaporated in vacuo at 25 C. to' asmall volume, and the concentrated solution is poured into 50 ml. of icewater. The white solid which precipitates is recovered by filtration,washed with water and recrystallized from ethyl acetate to give16o-methyl-pregmane-3 oc-Ol-l 1,20-dione; V

A solution of 22 g. of 16a-methyl-pregnane-3a-ol-ll, 20-dione Zl-acetateand l g; of p-toluene-sulfonic acid in 250 ml. of acetic anhydride isheated at reflux under nitrogen for'a period of approximately 3 days. 7Two grams of potassium acetate (anhydrous) is added, and thevolatilesolvents are separated by distillation in vacuo. The residualmaterial is extracted with benzene, and the benzene extract is filteredto remove insoluble material. The benzene extracts are evaporated to avolume of 100 ml. and petroleum ether is added to the cloud point. Theresulting solution is adsorbed on 600 g. of acidwashed alumina; thealumina adsorbate is then washed The adsorbateis then eluted with 85:15petroleum-ether-ether mixture, and the first four liters of eluate iscollected, and evaporated to dryness 'in vacuo to give a mixture of nolacetate containing la-methyl -'17(20) pregnene-3u,20-diol-ll-one3,20-diacetate. This mixture of enolates, weighing approximately' 14 g;,is dissolved in 50 ml. of benzene, an excess of perbenzoic. acid isadded, and the mixture is kept tion until the benzene layer is free ofperbenzoic acid; the benzene layer is then washed with water untilneutral,

dried, and the solvent evaporated in vacuo to give a resultin mixture;This mixture is then heated at reflux, with stirring, for a period ofabout 16 hours, and the reaction mixture is cooled, filtered, andtheinsoluble material is Washed with acetone. The filtered solution isevaporated in vacuo thereby removing the solvents, and the residualmaterial is slurried with water, and the aqueous mixture extracted withethyl acetate. The ethyl acetate extract is washed with water toneutrality, dried, and the solvent is evaporated in vacuo to give an'oil. This oil is crystallized'from' ether, and recrystallized fromethyl acetate-ether to give16a-methyl-pregnane-3a,17a,21-trioll1,20-dione' 2l-aceta'te. i '7 p p Asolution of 400mg. of l6m-methyl-pregnane=3a-17a, 2ltriol-ll,20-dione2l-acetatein 4 ml. of pyridine is added to the complex formed by theaddition of 400 mg. of chromium trioxide to 4 ml. of pyridine.- Themixture is swirled until thoroughly mixed, and then allowed to stand atroom temperature overnight. The reaction mixture is poured into water,and the aqueous mixture is extracted with ether, and then twice withethyl acetate. The combined ether and ethyl acetate extracts are washedwith dilute aqueous sulfuric acid at about 0 C., and thenwith wateruntil neutral. The organic solvent layer is then dried, the solvents areevaporated therefrom in vacuo, and the residual crystalline material ispurified by the crystallization from ethyl acetate to givel6ri-methyl-pr'egnane- 17:2,21-diOl-3 ,l l,20 trione" ZI-acetateJ To 100mg. of l6a-methyl-pregnane-l7a,2l-diol-3,l1, 20-trione ZI-a'cetatedissolved in 2 ml. of chloroform and 2.25 ml. of glacial acetic acid, ata temperature of 55 C., is added two drops of a 0.001; N solution of dryHBr in glacial. acetic acid. To about 0.38 ml. of 0.001 N HBr in glacialacetic acid, at 55 C., is added 0.43 nil. of a solution containing 40mg. of bromine in chloroform, and the resulting solution is added, overabout a 10-minute period, to the solution of the steroid, whilemaintaining the reaction mixture at about 55 C. The reaction mixture isallowed to stand at 55 C. for

. about one-half hour; a solution containing 250 mg. of

crystalline 'material, lGa-rnethyl-l7a,20-epoxy-pregnane-3a,30-diol-11-one 3,2.0-diacetate. The latter material is dissolved,without purification, in 200 m1. of methanol, 120 ml. of water and 10 g.of potassium bicarbonate, and theresulting solution is heated at refluxunder nitrogenfor a period of 16 hours. The methanol is evaporated fromthe hydrolysis solution in vacuo, and the residual oil is extracted fromthe resulting aqueous solution with chloroform. The chloroform extractis washed with water to neutrality, dried, and the chloroform isevaporated under reduced pressure. The residual oil is triturated withether, and the crystalline material thus formed is recrystallized fromethyl acetate-petroleum ether to give 16a-methyl-pregnane-3a,17a-diol-11,20 dione.

T o a solution of 7.0 g. of l6a-methyl pregnane-3a,17a-

diol-l1,20-dione in ml. of chloroform is added drop- 50:50ether-petroleum ether-mixtureto give about 5 g.. of

diol-ll,20-dione is mixedwith 5.0g, of anhydrous potas sium acetate, 4.0g. of sodium iodide and 0.03 ml. of

glacial acetic acid, and 100 m1. of acetone is added to the rated todryness in vacuo.

crystallized from methanol andrecrystallize'd'from ethyl.

sodium acetate in 3 ml. of water is added, and the resulting mixture isstirred for. about 5 minutes. Five milliliters of water are then added,and the aqueous mixture is extracted with ethyl acetate. The ethylacetate extract is washed with aqueous sodium bicarbonate soluiton toneutrality, then with water, dried, and the solvent is evaporated invacuo. The residual material is dissolvedin 2 ml. of acetone, and to thesolution is added 25 mg. of sodium bromide and 1 ml. of water. Theresulting mixture is heated under reflux for a period of about 5 hours,the reaction mixture is cooled, and the acetone is evaporated in vacuo.The residual material is extracted into ether, the ether extract iswashed with water, dried, and the. solvent is evaporated to a volume ofabout 1 ml.; petroleum ether is added to this solution, and thecrystallinematerial which separates is recovered and dried to giveapproximately mg. of 4-bromo 16a: methyl-pregnane lhl1-diol-3,11,20-trione 21- acetate,

A mixture of 33 mg. of. semicarbazide, 90 mg. of 4- bromo 16ozmethyl-pregnane-'l7m,21-diol=3,11,20-trione 'ZI-acetate, 90 mg. ofanhydroussodium sulfate, 0.3 m1.

of dimethyl formamide and 3.5 ml. of chloroform is heated under refluxin contact with a nitrogen atmosphere for a period of about 45 minutes.Three. milliliters of water are added to the reaction mixture and theresulting mixtureis heated under. reflux for about 10 minutes. The,mixture is cooled, extractedwith ethyl acetate and the washed and driedethyl acetate extract evapo- The residual material is acetate to givel6a-methyl-4-pregnene-1-7a,2l;diol 3,11,20- trione 3-semicarbaZone.ZI-acetate; Ml, 2255228" C. (dec). Fifty milligrams of 1'6amethyl4-pregnene-17a,21- diol 3,11,2 0 trione-3esemicarbazone.21-acetate is. dis solved in a mixture of 1.0 cc. of benzene and 1.0 cc.of

1.1 N methanolic potassium hydroxide, and the solution is allowed tostand at room temperature for a period of about minutes. The solution isthen acidified with acetic acid, the benzene is evaporated in vacuo, andthe residual material is recrystallized from ethyl acetate to give 1600methyl-4-pregnene-17a,2l-diol-3,11,20-tri0ne-3, -bis-semicarbazone.

A solution of 90 mg. of1600-methyl-pregnene-17a,2ldiol-3,11,20-trione-3-semicarbazone, 1.0 ml.of pyruvic acid, 1.0 ml. of glacial acetic acid and 1.0 ml. of water isallowed to stand for 20 hours at room temperature. The reaction solutionis poured into 8 ml. of water and the aqueous mixture is extracted with40 ml. of chloroform in six portions. The combined chloroform extract iswashed with an aqueous solution of sodium bicarbonate, dried over sodiumsulfate and evaporated in vacuo. The residual material is crystallizedfrom acetone to give l6a-methyl-4-pregnene-17a,21-diol-3,11,20-trione.

Various changes and modifications may be made in carrying out thepresent invention Without departing from the spirit and scope thereof.Insofar as these changes and modifications are within the purview of theannexed claims, they are to be considered as part of our invention.

We claim:

1. A compound having the following formula:

wherein Y is a substituent selected from the group consisting of ketoand hydroxy, X is selected from the group consisting of hydrogen, bromoand fluoro, and R is selected from the group consisting of hydrogen,lower hydrocarbon carbonyl and phosphoryl.

2. 60,160 dimethyl 4-pregnene-17a,21-diol-3-11,20- trione.

3. 60,1600 ethyl 4-pregnene-17a,2l-diol-3,11,20- trione ZI-phosphate.

4. 600,160: dimethyl 4-pregnene-l7a,21-diol-3,11,20- trione 21-acetate.

5. 600,160 dimethyl 4-pregnene-17a,21-diol-3,11,20- trione2l-(tertiary-butyl acetate).

6. 600,160 dimethyl 4-pregnene-11;9,170c,2l-triol-3,20 dione.

7. 60,160 dimethyl 4 -pregnene 11fi,17a,2l-triol-3, 20-dione 2l-acetate.

8. 60,160 dimethyl-4-pregnene-1lfi,17a,21-triol-3,20- dione 21-(tertiary-butyl acetate).

9. 600,160: dimethyl 4-pregnene-l15,170,21-t1i01-3, 20-dione21-phosphate.

10. 600,160: dimethyl-90t-fluoro-4-pregnene-1118,170,21-triol-3,20-dione.

11. 600,160 dimethyl 900-fluoro-4-pregnene-1118,170, 21triol-3,20-dioneill-acetate.

12. 600,160: dimethyl 900 fiuoro-4-pregnene-l7a,21- diol-3 ,11,20-trione.

22 13. 600,160; dimethyl-9a-fluoro-4-pregnene-170:,21-di0l-3,11,20-trione 21-acetate.

14. A compound having the following formula:

CHzOR wherein Y is a substituent selected from the group consisting ofketo and hydroxy, X is selected from the group consisting of hydrogen,bromo and fluoro, and R is se lected from the group consisting ofhydrogen, lower hydrocarbon carbonyl and phosphoryl.

15. 602,160 dimethyl-1,4-pregnadiene-17a,21-dio1-3,11, 20-tn'one.

16. 600,160: dimethyl-l,4-pregnadiene-1700,21-diol-3,11, 20-trione2l-phosphate.

17. 60,1600 dimethyl-1,4-pregnadiene-17a,21-diol-3,11, 20-trione21-acetate.

18. 600,160 dimethyl-l,4-pregnadiene-17a,2l-diol-3,l1, 20-trione21-(tertiary-butyl acetate).

19. 60:,160: dimethyl-1,4-pregnadiene-11fi,170z,21-t1'i01- 3,20-dione.

20. 600,160: dimethyl-l,4-pregnadiene-l lB,17a,21-triol- 3,20-dione21-acetate.

21. 600,160: dimethyl-lA-pregnadiene-l1B,l7u,21-t1'iol- 3,20-dione2l-tertiary-butyl acetate).

22. 60,160: dimethyl-1,4-pregnadiene-1113,1700,21-triol 3,20-di0ne21-phosphate.

23. 600,160: dimethyl 90z-fluoro-L4-pregnadiene-11B,1700,21-triol-3,20-dione.

24. 60,160 dirnethyl-90c-fiuoro-1,4-pregnadiene-11B,l700,21-triol-3,20-dione 2l-acetate.

25. 60,160; dimethyl-9a-fluoro-1,4-pregnadiene-17a, 21-diol-3,11,20-trione.

26. 600,160 dimethyl 90-fiuoro-1,4-pregnadiene-17a,21-dio1-3,11,20-trione Zl-acetate.

References Cited in the file of this patent UNITED STATES PATENTS2,602,769 Murray et a1. July 8, 1952 2,649,402 Murray et al Aug. 18,1953 2,773,060 Levin et a1. Dec. 4, 1956 2,785,203 Sarett Mar. 12, 19572,789,117 Sarett Apr. 16, 1957 2,797,229 Rails June 25, 1957 2,809,967Poos et al. Oct. 15, 1957 2,813,108 Hanze Nov. 12, 1957 2,866,799 Beyleret al Dec. 30, 1958 OTHER REFERENCES Szpilfogel et al.: Rec. Trav.Chem., (1956), pp. 475-80.

Spero et al.: 78 J.A.C.S., 6213-14 (1956).

Spero et al.: 79 I.A.C.S., 1515-16 (1957).

1. A COMPOUND HAVING THE FOLLOWING FORMULA:
 14. A COMPOUND HAVING THEFOLLOWING FORMULA: